The focus of the project is to employ both novel genetic control circuits and extensive experimental analysis to position the redox energetics of the E. coli cell to the optimal physiological and biochemical state for production of chemicals operating in a microaerobic mode. There are many examples where the requirements of rapid cell growth rate and highly efficient energy production from NADH utilization by the electron transport chain conflict with the demands of oxygen and NADH requiring reactions for the desired role of the cell as a specific biocatalyst. The positive aspect of oxygen reaction with reductant through the electron transport chain is apparent in a faster growth rate and improved protein synthesis and response to stressful conditions. The use of monooxygenases and other enzymes that require oxygen and a recycling reductant (NADH or NADPH) to form a chemical of commercial interest is growing with the characterization of many useful enzymes. Examples include specific oxygenases used in antibiotic synthesis, cytochrome P450's used in drug metabolic studies and chemical synthesis, BVMO enzymes that can synthesize esters and lactones, and enzymes involved in formation of natural flavonoid compounds. Thus there is a growing need for improved understanding and engineering of cells poised at the optimum condition for efficient production of these compounds. We will engineer cells that have an optimized window of function of the electron transport system allowing suitable growth and cell energetics while allowing a high portion of the NADH to be used in the desired biocatalyst mode for product formation. For feedback control of individual proteins required for ETS function, we will express an essential gene under control of the Arc system such that it is only on as the cells become more aerobic, while at the same time expressing another gene essential for proper ETS function under a different control system so it exhibits a feedback response evoking a defined functional level of ETS in a prescribed manner as oxygen and culture conditions are changed. Once a series of constructs are made with altered ETS levels and response patterns, preliminary culture studies will be conducted to see which are most likely to be useful in practical applications. Those strains selected for more extensive defined bioreactor studies will be examined for patterns of gene expression and metabolite formation under conditions of various oxygen levels and other culture parameters. The most characterized strains will then be subjected to studies of a more practical context. In this segment of the proposal two situations common to industrial processes will be used: one which involves the use of non-growing whole cells in a biocatalyst mode where the formation of a cell mass with optimal productivity, and the other which will examine the simultaneous growth and production mode and will optimize both the growth rate and the overall volumetric productivity of the desired product. The project will be carried out under the direction of a microbiologist and biochemist (G.N. Bennett) with expertise in microbial physiology and genetics;and a bioengineer (K.-Y. San) with expertise in metabolic engineering, bioprocess optimization, and flux analysis.